Chiral columns with broad chiral selectivity

ABSTRACT

A general chiral column with a multipleproline-based chiral stationary phase. Embodiments include chiral stationary phases of the following formula:  
                 
 
     wherein n is any integer of 2 or greater, and analogs and isomers thereof.

PRIORITY

[0001] This application claim priority to U.S. Application No.60/465,930, filed Apr. 28, 2003, the contents of which are incorporatedherein by reference in their entirety.

GOVERNMENT SUPPORT

[0002] This invention was made in connection with Grant Numbers NIH 1R01 GM63812-01 and NIH 1 R01 GM60637-01A1, from the National Institutesof Health. The Government has rights to this invention.

FIELD OF THE INVENTION

[0003] The present invention relates to the field of chiral chemistry.More particularly, the present invention relates to the separation ofenantiomers, i.e., those isomers in which the arrangement of atoms orgroups is such that the two molecules are not superimposable.

[0004] The present inventor has developed a new class of chiral columnsthat can resolve a large number of racemic compounds. These columns arestable and can be used with a number of mobile phase solvents.

BACKGROUND OF THE INVENTION

[0005] Stereoisomers are those molecules which differ from each otheronly in the way their atoms are oriented in space. Stereoisomers aregenerally classified as diastereomers or enantiomers; the latterembracing those which are mirror-images of each other, the former beingthose which are not. The particular arrangement of atoms thatcharacterize a particular stereoisomer is known as its opticalconfiguration, specified by known sequencing rules as, for example,either + or − (also D or L) and/or R or S.

[0006] Though differing only in orientation, the practical effects ofstereoisomerism are important. For example, the biological andpharmaceutical activities of many compounds are strongly influenced bythe particular configuration involved. Indeed, many compounds are onlyof widespread utility when employed in a given stereoisomeric form.

[0007] Living organisms usually produce only one enantiomer of a pair.Thus only (−)-2-methyl-1-butanol is formed in yeast fermentation ofstarches; only (+)-lactic acid is formed in the contraction of muscle;fruit juices contain only (−)-malic acid, and only (−)-quinine isobtained from the cinchona tree. In biological systems, stereochemicalspecificity is the rule rather than the exception, since the catalyticenzymes, which are so important in such systems, are optically active.For example, the sugar (+)-glucose plays an important role in animalmetabolism and is the basic raw material in the fermentation industry;however, its optical counterpart, or antipode, (−)-glucose, is neithermetabolized by animals nor fermented by yeasts. Other examples in thisregard include the mold Penicillium glaucum, which will only consume the(+)-enantiomer of the enantiomeric mixture of tartaric acid, leaving the(−)-enantiomer intact. Also, only one stereoisomer of chloromycetin isan antibiotic; and (+)-ephedrine not only does not have any drugactivity, but it interferes with the drug activity of its antipode.Finally, in the world of essences, the enantiomer (−)-carvone providesoil of spearmint with its distinctive odor, while its opticalcounterpart (+)-carvone provides the essence of caraway.

[0008] Thus, as enzymes and other biological receptor molecules possesschiral structures, enantiomers of a racemic compound may be absorbed,activated, and degraded by them in different manners. This phenomenoncauses that in many instances, two enantiomers of a racemic drug mayhave different or even opposite pharmacological activities. In order toacknowledge these differing effects, the biological activity of eachenantiomer often needs to be studied separately. This and other factorswithin the pharmaceutical industry have contributed significantly to theneed for enantiomerically pure compounds and thus the need for chiralchromatography.

[0009] Accordingly, it is desirable and oftentimes essential to separatestereoisomers in order to obtain the useful version of a compound thatis optically active.

[0010] Separation in this regard is generally not a problem whendiastereomers are involved: diastereomers have different physicalproperties, such as melting points, boiling points, solubilities in agiven solvent, densities, refractive indices etc. Hence, diastereomersare normally separated from one another by conventional methods, such asfractional distillation, fractional crystallization or chromatography.

[0011] Enantiomers, on the other hand, present a special problem becausetheir physical properties are identical. Thus they cannot as a rule—andespecially so when in the form of a racemic mixture—be separated byordinary methods: not by fractional distillation, because their boilingpoints are identical; not by conventional crystallization because(unless the solvent is optically active) their solubilities areidentical; not by conventional chromatography because (unless theadsorbent is optically active) they are held equally onto the adsorbent.The problem of separating enantiomers is further exacerbated by the factthat conventional synthetic techniques almost always produce a mixtureof enantiomers. When a mixture comprises equal amounts of enantiomershaving opposite optical configurations, it is called a racemate;separation of a racemate into its respective enantiomers is generallyknown as a resolution, and is a process of considerable importance.

[0012] Chiral columns that can resolve a large number of racemiccompounds (general chiral columns) are in high demand. They are neededroutinely in many laboratories, especially in pharmaceutical industry.Prior to the present invention, Daicel columns, macrocyclic antibioticcolumns, and the Whelk-O columns were probably known as the industrialleaders in this type of general chiral columns. The present inventor hasdeveloped a new class of general chiral columns based on the use ofproline and its analogues.

[0013] Furthermore, and importantly, the columns of the presentinvention have the capability of resolving at least a similar or higherpercentage of the compounds tested. Furthermore, the columns of thepresent invention provide better separation on some of the compoundstested and can resolve certain compounds that cannot be resolved withthe commonly used commercial columns listed above.

[0014] The columns of the present inventions are stable and can be usedwith a large number of mobile phase solvents. Therefore, the columns ofthe present invention should find important applications as generalchiral columns.

[0015] As stated above, a large number of chiral columns have beenprepared in the past; however, only a few demonstrated broad chiralselectivity. The successful examples include the popular Daicel columns,the Chirobiotic columns, and the Whelk-O½ columns. The Daicel columnsare prepared by coating sugar derivatives onto silica gel. Chirobioticcolumns are prepared by immobilizing macrocyclic glycopeptides ontosilica gel. Whelk-O ½ columns contain both electron rich and electrondeficient aromatics. These columns have broad chiral selectivity andhave been applied successfully to resolve a fair number of racemiccompounds. They have different selectivity and stability profiles. Theirselectivities complement each other in some cases, while they duplicateeach other in other cases. Some of the columns are more suited forreversed phase conditions and others for normal phase conditions. Eachcolumn has its own strengths and weaknesses. Despite these progresses,there are still many compounds that cannot be resolved or resolved wellusing these commercial available columns. Therefore, there is still asignificant need to develop new columns that have relatively broadchiral selectivity.

SUMMARY OF THE INVENTION

[0016] The present invention is directed to a chiral selector thatrepresents an improvement in the art of enantiomeric separation. Thus,one embodiment of the present invention is a general chiral column witha multiple proline-based chiral stationary phase.

[0017] Another embodiment of the present invention is a chiralstationary phase made of peptides with 2 or more prolines, includingchiral stationary phases with 2, 3, 4, 5, 6, or 10 prolines. Alsoincluded within the scope of the present invention are analogs andisomers of prolines, and analogs and isomers of the chiral selectorcompounds of the present invention.

[0018] Another embodiment of the present invention is a chiralstationary phase of the following formula:

[0019] wherein n is any integer of 2 or greater, and analogs and isomersthereof. Another embodiment of the present invention is where n is anyinteger from 2-10.

[0020] The separations achieved for analytes are comparable or superiorto those achieved on Daicel AD, Daicel OD, and Whelk O2 columns. Themultiple proline-based chiral stationary phases of the present inventionshow promise as a superior general chiral column.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021]FIG. 1 shows the structure for amino acid L-Proline and itsassociated stationary phases Fmoc-Pro₄-Silica (1) andFmoc-Pro-Pro-Silica (2).

[0022]FIG. 2 shows the synthesis of one embodiment of the presentinvention, Fmoc-Pro₄-(Me)Ahx-APS chiral stationary phase.

DESCRIPTION OF THE INVENTION

[0023] The present inventor has developed a new chiral column that hasrelatively broad chiral selectivity, when compared with Daicel columnsand Whelk O2 column, as industry standards or industry models.Additionally, the chiral columns of the present invention are stable ina number of mobile phase conditions.

[0024] The present inventor found from the same library a chiralselector [Fmoc-Hyp(tBu)-Pro] that has broad chiral selectivity whencompared to chiral selectors such as those mentioned above, which maylead to a general chiral column. Although this high throughput approachis designed to identify effective chiral selectors for a given analyte,newly prepared columns could be evaluated quickly for general chiralseparations.

[0025] In certain embodiments, this column contains only hydrogenbonding acceptor groups but no hydrogen bonding donor groups. It wassubsequently evaluated using racemic compounds that contain one or morehydrogen bonding donor groups (HO or NH group). Of the thirteencompounds chosen based on their availability, it resolved nine. Sincemany compounds contain one or more hydrogen bonding donor groups, thecolumn of the present invention could prove to be a useful generalchiral column.

[0026] The success rate of the chiral column of the present inventioncompares well with the best commercially available general chiralcolumns developed over the last few decades. According to the onlinecatalogue of Chiral Technologies, distributor of the most successfulchiral columns, their five best columns could resolve 80% of the racemiccompounds submitted to them.

[0027] Proline is a unique amino acid in many ways (FIG. 1). Instead ofhaving a primary amino group as in other α-amino acids, it contains asecondary amine. Because of the cyclic structure, rotation around thenitrogen-α-carbon bond is restricted. Also because of the cyclicstructure, proline is not ideally suited for α-helix or β-sheetconformation; instead, polyproline forms its own unique helicalconformation (Polyproline I and polyproline II). The amide bond inpolyproline is sterically hindered compared with other oligopeptides.The distinctly different conformational and structural features ofpolyprolines suggest that they may behave quite differently from othershort oligopeptides that have been studied in chiral chromatography.

[0028] The present inventors discovered that proline based chiralselectors, including the embodiment tetraproline based chiral stationaryphase 1 (FIG. 1) and diproline based chiral stationary phase 2 haverelatively broad chiral selectivity, while mono-pro stationary phase islargely ineffective.

[0029] Immobilization of the chiral selectors of the present inventionto silica gel is accomplished through a linker group. One example of alinker group of the present invention is a N-alkylamino group. A secondexample is a N-methylamino group. Another example is6-N-methylaminohexanoic acid. The particular linker group can beselected by on of ordinary skill in the art depending on the analyte tobe tested. For example, when the selector Fmoc-Pro-Pro is immobilizedusing 6-N-methylaminohexanoic acid, it may resolve about 16 out of about22 analytes tested. For the same chiral selector, when immobilized using6-amonihexanoic acid, it resolved about 4 out of the same group ofanalytes.

[0030] Preferred linkers of the present invention do not introduce ahydrogen-bonding donor NH group next to the Pro tetramer. The amide bondbetween this linker and pro residue is also more sterically hindered dueto the N-methyl group. A preferred linker of the present invention isshown in connection with the synthesis shown in FIG. 2.

[0031] Additionally, the stationary phase compounds of the presentinvention may comprise various end-capping groups as known in the art.

[0032] By use of the term proline with respect to the present invention,it is understood that analogs and isomers of proline are included. Forexample all stereoisomers are included. Additionally, analog areincluded. Examples of the analogs that are included herein are thosewith the following structure feature:

[0033] wherein n is an integer (such as 1, 2, 3, 4, 5, etc.) and X isO,S,N.

[0034] The columns of the present invention were evaluated using 22racemic mixtures chosen based on their availability. The separationfactors along with the capacity factors for the fast eluting enantiomerare shown below. For comparison, resolution of these 22 compounds wasalso studied with Daicel AD, Daicel OD, and Whelk O2 columns, the threemost popular chiral columns used in normal phase mode. Their separationfactors along with the capacity factors for the fast eluting enantiomerare also shown in an example, below. As seen in the examples, thedi-proline and tetra-proline columns of the present invention aresuperior.

[0035] These covalently bound columns of the present invention arestable in common organic solvents, including CH₂Cl₂ and CHCl₃.Therefore, a wide selection of mobile phase conditions could be appliedin method development. For several analytes, the present inventorattempted resolution with CH₂Cl₂/hexane as the mobile phase, findingcolumn efficiency.

[0036] In terms of potential interaction modes with the analytes,examples of the chiral selectors of the present invention are formingattractive hydrogen bonds with the analyte.

[0037] The following examples and experimental section are designed tobe purely exemplary in nature. Thus, this section should not be viewedas being limiting of the present invention.

EXAMPLES

[0038] Throughout this section, various abbreviations are used,including the following: DIC, diisopropylcarbodiimide; HATU,O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate; DIPEA, N,N-Diisopropylethylamine; DMF,N,N-Dimethylformamide; DCM, Dichloromethane; Fmoc-(Me)Ahx-OH,6-[(9H-fluoren-9-ylmethoxy)carbonyl]methylamino hexanoic Acid;Fmoc-Pro-OH, N-α-Fmoc-L-proline.

[0039] General Supplies and Equipment:

[0040] Amino acid derivatives were purchased from NovaBiochem (SanDiego, Calif.). All other chemicals and solvents were purchased fromAldrich (Milwaukee, Wis.), Fluka (Ronkonkoma, N.Y.), or FisherScientific (Pittsburgh, Pa.). HPLC grade Kromasil® silica gel (particlesize 5 μm, pore size 100 Å, and surface area 298 m²/g) was purchasedfrom Akzo Nobel (EKA Chemicals, Bohus, Sweden). Selecto silica gel(32-63 μm) from Fisher Scientific was used for flash columnchromatographic purification of target compounds. Thin-layerchromatography was completed using EM silica gel 60 F-254 TLC plates(0.25 mm; E. Merck, Merck KGaA, 64271 Darmstadt, Germany). Elementalanalyses were conducted by Atlantic Microlab, Inc. (Norcross, Ga.). HPLCanalyses were completed with a Beckman analytical gradient system(System Gold). UV spectra were obtained with a Shimadzu UV 201spectrometer (cell volume 3 mL; cell pass length 10 mm).

Example 1 Preparation of Chiral Stationary Phase Fmoc-Pro-(Me)Ahx-APS

[0041] To 0.80 g of (Me)Ahx-APS silica prepared previously (the surface(Me)Ahx concentration is 0.64 mmol/g) are added mixtures of Fmoc-Pro-OH(3 equiv., 0.52 g), HATU (3 equiv., 0.58 g), and DIPEA (3 equiv., 0.20g) in 8 mL of DMF. After agitating for 6 h, the resulting silica isfiltered and washed with DMF, Methanol, and DCM to yield the desiredchiral stationary phase. The surface Pro concentration is determined tobe 0.57 mmol/g based on the Fmoc cleavage method. The resulting chiralstationary phase is packed into a 50×4.6 mm HPLC column using a standardslurry packing method.

Example 2 Preparation of Chiral Stationary Phase Fmoc-Pro₂-(Me)Ahx-APS

[0042] To 0.80 g of (Me)Ahx-APS silica prepared previously (the surface(Me)Ahx concentration is 0.64 mmol/g) are added mixtures of Fmoc-Pro-OH(3 equiv., 0.52 g), HATU (3 equiv., 0.58 g), and DIPEA (3 equiv., 0.20g) in 8 mL of DMF. After agitating for 6 h, the resulting silica isfiltered and washed with DMF, Methanol, and DCM to yield the desiredchiral stationary phase. The surface Pro concentration is determined tobe 0.57 mmol/g based on the Fmoc cleavage method. The resulting chiralstationary phase was packed into a 50×4.6 mm HPLC column using thestandard slurry packing method.

Example 3 Preparation of Chiral Stationary Phase Fmoc-Pro₄-(Me)Ahx-APS

[0043] To 0.80 g of (Me)Ahx-APS silica prepared previously (the surface(Me)Ahx concentration is 0.64 mmol/g) were added mixtures of Fmoc-Pro-OH(3 equiv., 0.52 g), HATU (3 equiv., 0.58 g), and DIPEA (3 equiv., 0.20g) in 8 mL of DMF. After agitating for 6 h, the resulting silica isfiltered and washed with DMF, Methanol, and DCM to yield the desiredchiral stationary phase. The surface Pro concentration was determined tobe 0.57 mmol/g based on the Fmoc cleavage method. The resulting chiralstationary phase was packed into a 50×4.6 mm HPLC column using thestandard slurry packing method.

[0044] The following examples set forth various chromatographicmeasurements. Therein, retention factor (k) equals to (t_(r)-t₀)/t₀ inwhich t_(r) is the retention time and t₀ is the dead time. Dead time t₀was measured with 1,3,5-tri-t-butylbenzene as the void volume marker.Flow rate at 1 mL/min., UV detection at 254 nm.

Example 4

[0045] This example compares chromatographic resolution of racemiccompounds with chiral columns, including embodiments of the presentinvention (Pro2, Pro4, Pro6). k₁ is the retention factor of the leastretained enantiomer. This example also shows that a mono-proline chiralcolumn does not perform suffieiently.

[0046] Furthermore, this example shows embodiments of the presentinvention in comparison with known columns. Daicel Daicel Analyte nameAnalyte Structure Pro1 Pro2 Pro4 Pro6 OD AD Whelk-O2 Benzoin

α: 1 k₁: 5.78 3% IPA α: 1.07 k₁: 8.22 3% IPA α: 1.09 k₁: 6.35 3% IPA α:1.12 k₁: 16.0 5% IPA α: 1.61 k₁: 4.68 3% IPA α: 1.32 k₁: 3.46 15% IPA α:2.12 k₁: 1.83 5% IPA Hydrobenzoin

α: 1 k₁: 17.71 4% IPA α: 1.12 k₁: 21.15 4% IPA α: 1.13 k₁: 17.98 4% IPAα: 1.15 k₁: 25.79 8% IPA α: 1 k₁: 7.35 4% IPA α: 1.08 k₁: 5.15 8% IPA α:1.33 k₁: 4.18 4% IPA Benzoin oxime

α: 1 k₁: 12.28 20% IPA α: 1.09 k₁: 16.08 20% IPA α: 1.13 k₁: 15.36 20%IPA α: 1.20 k₁: 41.45 30% IPA α: 1.13 k₁: 2.82 10% IPA α: 1.24 k₁: 4.5515% IPA α: 1.31 k₁: 1.40 10% IPA 2,2,2-Trifluoro- (9-anthryl) ethanol

α: 1 k₁: 16.40 10% IPA α: 1.28 k₁: 23.44 10% IPA α: 1.56 k₁: 18.48 10%IPA α: 1.78 k₁: 22.58 25% IPA α: 1.13 k₁: 1.26 15% IPA α: 1.47 k₁: 1.9910% IPA α: 1.13 k₁: 0.62 10% IPA α-(pentafluoroethyl)-α-(trifluoromethyl)- Benzenemethanol

α: 1 k₁: 19.31 3% IPA α: 1.06 k₁: 16.08 3% IPA α: 1.10 k₁: 8.91 3% IPAα: 1.10 k₁: 8.62 5% IPA α: 1.16 k₁: 0.90 1% IPA α: 1.11 k₁: 0.79 3% IPAα: 1 k₁: 0.70 3% IPA Warfarin

α: 1 k₁: 13.91 10% IPA & 1% AcOH α: 1.11 k₁: 10.57 10% IPA & 1% AcOH α:1.08 k₁: 11.19 10% IPA & 1% AcOH α: 1.18 k₁: 17.50 10% IPA & 1% AcOH α:2.49 k₁: 6.40 15% IPA α: 3.94 k₁: 5.02 20% IPA α: 1.97 k₁: 10.06 20% IPAα-Methyl-2- Naphthalenemethanol

α: 1 k₁: 13.36 1% IPA α: 1 k₁: 22.36 1% IPA α: 1.04 k₁: 17.62 1% IPA α:1 k₁: 18.81 3% IPA α: 1 k₁: 6.25 3% IPA α: 1.05 k₁: 1.98 10% IPA α: 1.02k₁: 4.39 3% IPA 1-Acenaphthenol

α: 1 k₁: 7.83 3% IPA α: 1 k₁: 13.36 3% IPA α: 1 k₁: 10.28 3% IPA α: 1k₁: 28.06 3% IPA α: 1.16 k₁: 5.46 3% IPA α: 1.08 k₁: 6.58 3% IPA α: 1.28k₁: 4.96 3% IPA 3-Phenyl-Glycidol

α: 1 k₁: 5.23 3% IPA α: 1 k₁: 5.64 3% IPA α: 1 k₁: 6.77 3% IPA α: 1 k₁:10.38 5% IPA α: 1.15 k₁: 16.87 10% IPA α: 1 k₁: 8.01 8% IPA α: 1.37 k₁:8.74 10% IPA 1,1,′-Bi-2-naphthol

α: 1.04 k₁: 11.48 75% IPA α: 1.16 k₁: 32.80 75% IPA α: 1.29 k₁: 28.8375% IPA α: 1.42 k₁: 9.94 90% IPA α: 1.16 k₁: 4.49 8% IPA α: 1.13 k₁:3.58 25% IPA α: 1 k₁: 1.26 5% IPA 2,2′-Dihydroxy- 5,5′,6,6′,7,7′,8,8′-Octahydro-1,1′- binaphthyl

α: 1.14 k₁: 12.58 10% IPA α: 1.17 k₁: 11.10 10% IPA α: 1.32 k₁: 10.9510% IPA α: 1.67 k₁: 19.75 25% IPA α: 1.32 k₁: 3.98 5% IPA α: 1 k₁: 8.5310% IPA α: 1 k₁: 4.47 5% IPA 1,2,3,4-Tetrahydro-4- (4-methoxyphenyl)-6-methyl-2-thioxo- 5- pyrimidinecarboxylic acid ethyl ester

α: 1.05 k₁: 14.81 15% IPA α: 1.18 k₁: 18.68 15% IPA α: 1.24 k₁: 12.6015% IPA α: 1.20 k₁: 23.53 15% IPA α: 1.15 k₁: 2.13 15% IPA #α: 1.40 k₁:3.44 15% IPA α: 1.16 k₁: 2.27 15% IPA 1,2,3,4-Tetrahydro-4-(4-hydroxyphenyl)- 6-methyl-2-thioxo-5- Pyrimidinecarboxylic acid ethylester

α: 1.12 k₁: 44.66 30% IPA α: 1.20 k₁: 27.80 50% IPA α: 1.41 k₁: 25.0730% IPA α: 1.32 k₁: 30.13 70% IPA α: 1.30 k₁: 2.87 15% IPA #α: 1.36 k₁:4.62 15% IPA α: 1 k₁: 2.23 15% IPA 1-[1,2,3,4- Tetrahydro-4-(4-methoxyphenyl)- 6-methyl-2-thioxo- 5- pyrimidine[ethanone

α: 1 k₁: 27.03 15% IPA α: 1.20 k₁: 40.60 15% IPA α: 1.21 k₁: 25.72 15%IPA α: 1.34 k₁: 33.90 25% IPA α: 1.18 k₁: 2.62 15% IPA #α: 1.70 k₁: 3.6215% IPA α: 1 k₁: 1.83 15% IPA Hexobarbital

α: 1 k₁: 28.86 1% IPA α: 1 k₁: 22.28 1% IPA α: 1 k₁: 16.98 1% IPA α: 1k₁: 11.26 3% IPA α: 1.12 k₁: 6.26 5% IPA α: 1.46 k₁: 2.42 8% IPA α: 1 #k₁: 1.95 5% IPA Temazepam

α: 1.09 k₁: 22.03 2% IPA α: 1 k₁: 25.54 2% IPA α: 1 k₁: 20.24 2% IPA α:1 k₁: 25.52 5% IPA α: 1 k₁: 3.39 25% IPA α: 1 k₁: 4.12 25% IPA α: 1.19 #k₁: 3.40 25% IPA 5-Methyl- 5-(2,5-dichloro phenyl)hydantoin

α: 1 k₁: 11.21 15% IPA α: 1.16 k₁: 14.6 15% IPA α: 1.17 k₁: 8.55 15% IPAα: 1.34 k₁: 11.64 25% IPA α: 1.08 k₁: 4.29 8% IPA α: 1 k₁: 4.80 8% IPAα: 1.11 # k₁: 1.96 10% IPA 5-Methyl-5-phenyl hydantoin

α: 1 k₁: 16.24 8% IPA α: 1.10 k₁: 25.00 8% IPA α: 1.15 k₁: 15.6 8% IPAα: 1.32 k₁: 10.7 20% IPA α: 1.09 k₁: 4.06 8% IPA α: 1 k₁: 3.24 8% IPA α:1.46 # k₁: 1.67 10% IPA Mephenytoin

α: 1 k₁: 5.86 2% IPA α: 1.14 k₁: 7.86 2% IPA α: 1.14 k₁: 6.93 2% IPA α:1.27 k₁: 9.85 3% IPA α: 1.10 k₁: 5.20 4% IPA α: 1.37 k₁: 3.56 4% IPA α:1 # k₁: 3.35 4% IPA sec-Butyl-carbanilate

α: 1 k₁: 3.62 1% IPA α: 1 k₁: 7.30 1% IPA α: 1 k₁: 7.64 1% IPA α: 1 k₁:15.34 3% IPA α: 1 k₁: 6.18 2% IPA α: 1.04 k₁: 3.35 2% IPA α: 1.05 # k₁:3.36 1% IPA Methyl Mandelate

α: 1.02 k₁: 7.97 1% IPA α: 1.10 k₁: 10.8 1% IPA α: 1.17 k₁: 8.31 1% IPAα: 1 k₁: 17.49 3% IPA α: 1.25 k₁: 2.82 3% IPA α: 1.08 k₁: 1.85 10% IPAα: 1.07 # k₁: 1.12 10% IPA

Example 5 Specific Embodiments, for Exemplary Purposes, of theStationary Phase Compounds of the Present Invention and Silica Supports

[0047] This example sets forth poly-proline compounds of the presentinvention, including embodiments with different end-capping groups. Theend-capping groups are bonded to the nitrogen atom that is further awayfrom the support. As is noted in the example, some end-capping groupssuch as pivaloyl (PIV) (CSP-1) are more effective for some analytes thanothers, such as TAPA. Overall, several different end-capping groupsuseable with the present invention such as Piv, Fmoc, Boc, Cbz, Aca,Dmb, Tpa all work well. CSP-11, which has not end-capping group, did notperform as well with respect to some analytes.

Example 6

[0048] This example compares chromatographic resolution of racemiccompounds with Fmoc-Pro-Pro-Pro-Pro-N(Me)-Ahx-APS, which is anembodiment of the present invention, in two mobile phase systems.Accordingly, this example helps demonstrate the flexibility of chiralstationary phases of the present invention in different mobile phasesystems. DCM/Hex/ Analyte name IPA/Hex MeOH Benzoin α: 1.09 α: 1.07 k₁:6.35 k₁: 11.61 3% IPA 5% DCM in Hexane Hydrobenzoin α: 1.13 α: 1.12 k₁:17.98 k₁: 12.93 4% IPA 40% DCM in Hexane Benzoin oxime α: 1.13 α: 1.08k₁: 15.36 k₁: 15.85 20% IPA 100% DCM 2,2,2-Trifluoro-1- α: 1.56 α: 1.20(9-anthryl) k₁: 18.48 k₁: 9.54 ethanol 10% IPA 100% DCMa-(pentafluoroethyl)- α: 1.10 α: 1.06 a-(trifluoromethyl)- k₁: 8.91 k₁:28.23 Benzenemethanol 3% IPA 30% DCM in Hexane Warfarin α: 1.08 α: 1 k₁:11.19 k₁: 5.84 10% IPA & 1% 30% DCM AcOH in Hexane& 1% AcOHSec-Phenethyl α: 1.02 α: 1.02 alcohol k₁: 8.07 k₁: 13.08 1% IPA 10% DCMin Hexane α-Methyl-2- α: 1.04 α: 1 Naphalenemethanol k₁: 17.62 k₁: 23.661% IPA 10% DCM in Hexane 1-Acenaphthenol α: 1 α: 1 k₁: 10.28 k₁: 7.31 3%IPA 30% DCM in Hexane 3-Phenyl-Glycidol α: 1 α: 1 k₁: 6.77 k₁: 7.39 3%IPA 30% DCM in Hexane 1,1′-Bi-2-naphthol α: 1.29 α: 1.06 k₁: 23.83 k₁:12.21 75% IPA 1% MeOH in Hexane 2,2′-Dihydroxy- α: 1.32 α: 15,5′,6,6′,7,7′,8,8′- k₁: 10.95 k₁: 12.08 Octahydro-1,1′- 10% IPA 50% DCMbinaphthyl in Hexane 1,2,3,4-Tetrahydro-4- α: 1.24 α: 1.18(4-methoxyphenyl)- k₁: 12.60 k₁: 6.26 6-methyl-2-thioxo- 15% IPA 60% DCM5-pyrimidinecarboxylic acid in Hexane ethyl ester 1,2,3,4-Tetrahydro-4-α: 1.41 α: 1.19 (4-hydroxyphenyl)- k₁: 25.07 k₁: 12.726-methyl-2-thioxo-,5- 30% IPA 3% MeOH Pyrimidinecarboxylic acid inHexane ethyl ester 1-[1,2,3,4-Tetrahydro-4- α: 1.21 α: 1.22(4-methoxyphenyl)- k₁: 25.72 k₁: 12.20 6-methyl-2-thioxo- 15% IPA 60%DCM 5-pyrimidinyl]ethanone in Hexane Hexobarbital α: 1 α: 1 k₁: 16.98k₁: 8.08 1% IPA 30% DCM in Hexane Temazepam α: 1 α: 1 k₁: 20.24 k₁: 4.622% IPA 10% DCM in Hexane 5-Methyl- α: 1.17 α: 1.12 5-(2,5-dichloro) k₁:8.55 k₁: 13.56 phenylhydantoin 15% IPA 100% DCM 5-Methyl-5-phenyl α:1.15 α: 1.11 hydantoin k₁: 15.6 k₁: 18.51 8% IPA\ 100% DCM Mephentoin α:1.14 α: 1.17 k₁: 6.93 k₁: 17.10 2% IPA 20% DCM in Hexane sec-Butylcarbanilate α: 1 α: 1.12 k₁: 7.64 k₁: 4.16 1% IPA 10% DCM in HexaneMethyl Mandelate α: 1.17 α: 1 k₁: 8.31 k₁: 8.18 1% IPA 10% DCM in Hexane

[0049] The invention being described, it will be apparent to thoseskilled in the art that various modifications and variations can be madein the present invention without departing from the scope or spirit ofthe invention. Other embodiments of the invention will be apparent tothose skilled in the art from consideration of the specification andpractice of the invention disclosed herein. It is intended that theAttachments be considered as exemplary only, and not intended to limitthe scope and spirit of the invention.

[0050] Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,experimental results, and so forth used in the Specification andAttachments are to be understood as being modified by the term “about.”Accordingly, unless specifically indicated to the contrary, areapproximations that may vary depending upon the desired propertiessought to be obtained by the present invention.

References

[0051] The following references are incorporated by reference in theirentirety.

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I claim:
 1. A chiral stationary phase of the following formula:

wherein n is any integer of 2 or greater, and analogs and isomersthereof.
 2. The chiral stationary phase compound of claim 1, wherein nis any integer from 2 to
 10. 3. The chiral stationary phase compound ofclaim 1, wherein the support is a silica support.
 4. The chiralstationary phase compound of claim 1, wherein the linker is anN-alkylamino group.
 5. The chiral stationary phase compound of claim 4,wherein the linker is an N-methylamino group.
 6. The chiral stationaryphase compound of claim 4, wherein the linker is a 6-methylaminohexanoicacid group.
 7. The chiral stationary phase of a compound of claim 1,wherein the end-capping group is a Piv, Fmoc, Boc, Cbz, Aca, Tapa, Dmb,or a Tpa.
 8. The chiral stationary phase of claim 1, wherein the linkeris of the following formula:

wherein n is an integer from 1-10.
 9. The chiral stationary phasecompound of claim 1, wherein the linker is of the following formula:


10. The chiral stationary phase compound of claim 1, wherein theend-capping group is a Piv, Fmoc, Cbz, Aca, Tapa, Dub, or Tpa group. 11.The chiral stationary phase compound of claim 1, of the followingformula:


12. An optically active proline derivative of the formula:

wherein n is any integer of 2 or greater, and analogs and isomersthereof.
 13. A process for separating enantiomeric mixtures by liquidchromatography, comprising: providing a racemic mixture; providing achiral column that comprises an optically active multi-proline compoundor an analog or isomer thereof; and introducing the mixture to thechiral column.
 14. The process of claim 13, wherein the optically activemulti-proline compound is of the following formula:

wherein n is any integer of 2 or greater, and analogs and isomersthereof.
 15. The process of claim 13, wherein the optically activemulti-proline compound os of the following formula:

and analogs and isomers thereof.
 16. A chiral column, comprising achiral stationary phase compound of claim 1.